Ligand Distributions in Agarose Particles as Determined by Confocal Raman Spectroscopy and Confocal Scanning Laser Microscopy

Abstract

Confocal Raman spectroscopy and confocal scanning laser microscopy have been used to analyze ligand distributions within individual chromatographic adsorbent particles. Three different types of particles have been investigated. The first type was synthesized to have a uniform distribution of allyl groups, whereas the two others were designed to have a surface layer of sulphopropyl groups and cores containing allyl groups and dextran, respectively. With confocal Raman spectroscopy it was possible to follow the distribution of both the surface layer and the interior. The distribution of sulphopropyl groups was evaluated with both confocal scanning laser microscopy and confocal Raman spectroscopy, whereas the distributions of allyl groups and dextran were evaluated only with the latter method. The results from the confocal measurements showed the expected result with a uniform distribution of allyl groups in the first type of particle and surface layers of sulphopropyl groups and cores with dextran or allyl groups for the two others.

Confocal Raman Microscopy: Performance, Pitfalls, and Best Practice

“…Numerous workers have confirmed the benefits of using immersion objectives to depth profile complicated materials ranging from polymers and paper products to human skin. [29][30][31][32][33][34][35][36][37][38][39][40] As well as reducing the depth-scale compression, the oil has an additional beneficial effect of wetting out particle/air boundaries in porous, highly scattering particulate coatings, improving transparency and permitting confocal profiling of samples that would be opaque to a dry objective. 28 Nonetheless, a significant mismatch between the index of the coupling oil and the sample will result in a slight axial shift and distortion in the shape of individual layers, manifested by an attenuation that increases with depth.…”

Ion exchange chromatography of antibody fragments

“…Recently, confocal scanning laser microscopy has been introduced as an alternative and complementary method for studying protein adsorption at the matrix level (Dziennik et al, 2003;Hubbuch et al, 2003a,b;Linden et al, 1999Linden et al, , 2002Ljunglöf and Hjorth, 1996;Ljunglöf and Thömmes, 1998;Ljunglöf et al, 1999;Subramanian and Hommerding, 2005;Zhou et al, 2006). With this technique it is possible to study intra-particle protein and DNA concentrations, and also ligand distributions, directly within individual adsorbent particles (Larsson et al, 2002(Larsson et al, , 2003Ljunglöf et al, 2000). The high optical resolution (e.g., <1 mm) obtained with confocal microscopy allows visualization of the adsorption process after labeling of the protein molecules with a fluorescent probe.…”

A model for interpreting depth profiles of confocal Raman measurements in reflective and transmitting materials

“…Grauw et al adapted this formalism and considered a Gaussian beam, instead of a spherical beam, for the image‐formation in the back‐focal plane, and accordingly obtained a Lorenzian profile of Raman intensity from a thin polystyrene film. For thick transparent samples, the effect of refractive index mismatch has been studied in several articles . Everall formulated the axial spreading of focus inside the sample between paraxial and oblique rays using a geometrical optic approach and provided an analysis of the Gaussian intensity distribution within the axially broadened depth‐of‐focus as a function of the starting position of the ray on the objective, that is, the pupil parameter.…”

“…[13][14][15] This particular problem is prevalent in high numerical aperture (NA) dry objective systems. Although index-matching immersion medium can alleviate this problem, [8,16,17] they are limited with respect to sample type. Due to their greater flexibility, dry objectives remain a commonly used type, despite the issue of focus broadening.…”

“…For thick transparent samples, the effect of refractive index mismatch has been studied in several articles. [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] Everall [8,20] formulated the axial spreading of focus inside the sample between paraxial and oblique rays using a geometrical optic approach and provided an analysis of the Gaussian intensity distribution within the axially broadened depth-of-focus as a function of the starting position of the ray on the objective, that is, the pupil parameter. Baldwin and Batchelder [21] analysed the mean focal position and depth-of-focus based on the axial intensity distribution within the broadened depth-of-focus.…”